1. Field Of The Invention
The development of specific binding assay techniques has provided extremely useful analytical methods for determining various organic substances of diagnostic, medical, environmental and industrial importance which appear in liquid mediums at very low concentrations. Specific binding assays are based on the specific interaction between the substance under determination herein referred to as the "analyte", and a binding counterpart thereof. Where one of the analyte and its binding counterpart is an antibody and the other is a corresponding hapten or antigen, the assay is known as an immunoassay.
In conventional specific binding assay techniques, a sample of the liquid medium to be assayed is combined with reagent systems of various compositions. Such compositions include a labeled conjugate comprising a binding component incorporated with a label. The binding component in the labeled conjugate interacts with other constitutents, if any, of the reagent system and the analyte in the medium under assay to form two species or forms of the labeled conjugate, a bound-species and a free-species. In the bound-species, the binding component, e.g., a hapten or antigen, in the labeled conjugate is bound by a corresponding binding counterpart, e.g., an antibody, whereas in the free-species, the binding component is not so bound. The relative amount or proportion of the labeled conjugate that results in the bound-species compared to the free-species is a function of the presence or amount of the analyte in the test sample.
Where the labeled conjugate in the bound-species is essentially indistinguishable in the presence of the labeled conjugate in the free-species by the means used to monitor the label, the bound-species and the free-species must be physically separated in order to complete the assay. This type of assay is referred to in the art as "heterogeneous". Where the bound-species and free-species forms of the labeled conjugate can be distinguished in the presence of each other, a "homogeneous" format can be followed and the separation step avoided.
This invention relates to specific binding assay methods and reagent systems for the quantitative or qualitative determination of an analyte in a liquid medium. In particular, the present invention relates to such methods and systems, especially of the homogeneous type, wherein the label employed is an antienzyme, e.g., an inhibitory antibody, or fragment thereof, for an enzyme.
2. Description Of The Prior Art
The first highly sensitive specific binding assay to be discovered was the radioimmunoassay which employs a radioactive isotope as the label. Such an assay necessarily must follow the heterogeneous format since the monitorable character of the label is the same in the free- and bound-species. Because of the inconvenience and difficulty of handling radioactive materials and the necessity of a separation step, homogeneous assay systems have been devised using materials other than radioisotopes as the label component, including enzymes, bacteriophages, metals and organometallic complexes, coenzymes, enzyme substrates, enzyme modulators, e.g., activators and inhibitors, cycling reactants, spin radicals, organic and inorganic catalysts, prosthetic groups, chemiluminescent reactants, and fluorescent molecules.
Generally representative of such homogeneous specific binding assays are those described in the following references: U.S. Pat. Nos. 4,134,792; 4,226,978; 4,230,797; 4,238,195; 4,238,565; 3,935,074; 4,208,479; 4,233,401; 4,256,834; 3,817,837; 4,043,872; 3,996,345; 4,233,402; 4,160,645; 3,690,834; and 4,278,866; and British Pat. Specification No. 1,595,101. Of these techniques, the following involve, in some fashion, label monitoring reactions based in modulation of enzyme activity by anti-enzyme.
U.S. Pat. Nos. 4,134,792 and 4,278,866 and British Pat. Specification No. 1,595,101 describe specific binding assays employing an enzyme modulator as the label. When performed in the homogeneous mode, the modulation effect of the labeled conjugate on the enzyme, in most cases an inhibition of enzyme activity, is altered, usually decreased, in the bound-species.
U.S. Pat. Nos. 4,208,479 and 4,233,401 describe homogeneous specific binding assays wherein an enzyme is employed as the label. A labeled conjugate is constructed such that the catalytic activity of the labeling enzyme is substantially retained; however, upon binding of the binding counterpart, e.g., antibody to the labeled conjugate, enzymatic activity is diminished.
The use of anti-enzyme labels in specific binding assays, particularly of the homogeneous type, is described in U.S. patent application Ser. No. 285,605, filed July 21, 1981, and assigned to Miles Laboratories, Inc., Elkhart, Ind., USA, the parent company of the present assignee. Such patent application describes the use of antibodies to a variety of different enzymes as labels in specific binding assays and provides a particular example wherein anti-peroxidase is used as the label. The use of anti-peroxidase labels is also described by a former co-worker with the inventor U.S. of Ser. No. 285,605 in FEBS Letters 116(2): 285-288 (July 1980)-Ngo and Lenhoff.